PROJECT SUMMARY Retinal ganglion cells (RGCs) from each eye extend their axons ipsi- and contralaterally to the brain to establish the circuit for binocular vision. The lack of appropriate sidedness of RGC projections is debilitating in genetic disorders such as albinism, in which hypopigmentation of the retinal pigment epithelium (RPE) is linked to optic nerve misrouting and therefore altered stereo vision. Progress on this grant includes the identification of transcriptional regulators of the specification and differentiation of ipsi and contra RGCs; demonstration of the ventral ciliary margin zone (CMZ) as a source of ipsi RGCs; expression of the cell cycle regulator Cyclin D2 in the ventral CMZ; and dependence of the ipsi RGC projection on Cyclin D2. In the albino retina, these processes and the cellular integrity of the RPE, are disrupted, making the albino an excellent comparative model for the proposed studies. Here we explore a novel role for the CMZ in neural retinal development and RGC specification, and seek to uncover potential signaling networks for establishing proper RGC connections. We propose to combine studies of neurogenesis and fate mapping with transcriptomics of the CMZ, RPE, and neural retina to gain mechanistic insight into how loss of melanin in the RPE of albino animals causes a shift in cell fate from ipsi to contra during the establishment of the binocular circuit. To this end, we will establish a role for Cyclin D2 in the pace of the cell cycle, plane of cell division, and migration from the CMZ to the neural retina (Aim 1); study signaling between the RPE and CMZ, starting with the Wnt pathway, as a route to regulate RGC fate (Aim 2); identify the transcriptional networks that regulate RGC cell fate by performing single-cell RNA-Seq of the CMZ, neural retina, and RPE (Aim 3). Throughout, we will compare albino and pigmented retina. We hypothesize that events in the CMZ control timing of neurogenesis, which specifies RGC projection fate and that factors in the RPE provide directives to the CMZ and neural retina for ipsi/contra RGC fate acquisition. Significance: Probing early neural retinal development and parsing regulators of neurogenesis and cell fate emanating from the CMZ and RPE offer a novel mechanistic entry point to the long-standing enigma of how pigment and the RPE influences cell fate and RGC axon segregation at the optic chiasm. Identifying gene programs on how ipsi/contra RGC diversity arises reveals how decussating systems such as the binocular circuit are established. Such information is critical for driving stem cells into RGCs for replacement therapy and directing axon regeneration in injured and degenerating visual pathways.